scholarly journals Site‐directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote

2016 ◽  
Vol 60 (2) ◽  
Author(s):  
E. Wyroba ◽  
P. Kwaśniak ◽  
K. Miller ◽  
K. Kobyłecki ◽  
M. Osińska 
Keyword(s):  
Confocal Imaging ◽  
Site Directed Mutagenesis ◽  
Double Immunofluorescence ◽  
Subcellular Targeting ◽  
In Vivo Electroporation ◽  
Post Translational Modifications ◽  
Peptide Map ◽  
Point Mutagenesis

<p>Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post‐translational modifications (PTM) in proper targeting of <em>Paramecium</em> Rab7b paralogue – distinct from that of Rab7a directly involved in phagocytosis ‐ was studied using point mutagenesis, proteomic analysis and double immunofluorescence after <em>in vivo</em> electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P<sup>32</sup>] by 37.4% and of 32 [<sup>C14</sup><sup>–</sup>]UDP‐glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non‐mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non‐ mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.6<sup>3+</sup> characteristic for the glycan group attached to Thr200 in Rab7b using nano LC‐MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P<sup>32</sup>]and  [C<sup>14</sup><sup>‐</sup>]UDP‐ glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C‐region of Rab7b in <em>Paramecium</em> is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.   </p>

Distinct functional contributions of 2 GABP–NRF-2 recognition sites within the context of the human TOMM70 promoter

2006 ◽  
Vol 84 (5) ◽  
pp. 813-822 ◽  
Author(s):  
José R. Blesa ◽  
José Hernández-Yago
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Outer Mitochondrial Membrane ◽  
Mobility Shift ◽  
A Subunit ◽  
Expression Evidence ◽  
Electrophoretic Mobility Shift Assays

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP–NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP–NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP–NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

scholarly journals USP52 regulates DNA end resection and chemosensitivity through removing inhibitory ubiquitination from CtIP

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ming Gao ◽  
Guijie Guo ◽  
Jinzhou Huang ◽  
Jake A. Kloeber ◽  
Fei Zhao ◽  
...  
Keyword(s):  
Parp Inhibition ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Post Translational Modifications ◽  
Regulatory Complexity ◽  
Dna End Resection ◽  
End Resection

Abstract Human C-terminal binding protein (CtBP)–interacting protein (CtIP) is a central regulator to initiate DNA end resection and homologous recombination (HR). Several studies have shown that post-translational modifications control the activity or expression of CtIP. However, it remains unclear whether and how cells restrain CtIP activity in unstressed cells and activate CtIP when needed. Here, we identify that USP52 directly interacts with and deubiquitinates CtIP, thereby promoting DNA end resection and HR. Mechanistically, USP52 removes the ubiquitination of CtIP to facilitate the phosphorylation and activation of CtIP at Thr-847. In addition, USP52 is phosphorylated by ATM at Ser-1003 after DNA damage, which enhances the catalytic activity of USP52. Furthermore, depletion of USP52 sensitizes cells to PARP inhibition in a CtIP-dependent manner in vitro and in vivo. Collectively, our findings reveal the key role of USP52 and the regulatory complexity of CtIP deubiquitination in DNA repair.

Seeing the trees in the forest: selective electroporation of adipocytes within adipose tissue

2004 ◽  
Vol 287 (3) ◽  
pp. E574-E582 ◽  
Author(s):  
James G. Granneman ◽  
Pipeng Li ◽  
Yuyan Lu ◽  
Jacqueline Tilak
Keyword(s):  
Adipose Tissue ◽  
Adrenergic Receptor ◽  
Fat Cells ◽  
Subcellular Targeting ◽  
In Vivo Electroporation ◽  
Brown Adipocytes ◽  
Adrenergic Signaling ◽  
Large Size ◽  
White Adipocytes

Electroporation has been recently adapted for the transfer of macromolecules into cells of tissues in vivo. Although mature adipocytes constitute <20% of cells residing in adipose tissue, we hypothesized that fat cells might be susceptible to selective electrotransfer of plasmid DNA owing to their large size relative to other cells in the tissue. Results demonstrate the feasibility of electroporating DNA into mature fat cells with >99% selectivity over other cells in the tissue. Further experiments used the “adiporation” technique to image the subcellular targeting of fluorescent bioreporter molecules to the nucleus, mitochondria, and lipid droplets of adipocytes within intact adipose tissue. Finally, we utilized fluorescent bioreporters to examine the effects of constitutive activation of the β-adrenergic signaling pathway in adipocytes. These results demonstrate that overexpression of rat β1-adrenergic receptors alters the cellular morphology of white adipocytes in a fashion that mimics the effects of systemic infusion of β3-adrenergic receptor agonists. Hallmarks of the altered morphology include pronounced fragmentation of the single lipid droplet, repositioning of the nucleus, and induction of mitochondrial biogenesis. These results indicate that activation of β-adrenergic signaling within adipocytes is sufficient to induce a phenotype that resembles typical brown adipocytes and suggest that in vivo electroporation will allow molecular dissection of the mechanisms involved.

scholarly journals Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

2016 ◽  
Vol 473 (11) ◽  
pp. 1605-1615 ◽  
Author(s):  
Sara R. Costa ◽  
Magdalena Marek ◽  
Kristian B. Axelsen ◽  
Lisa Theorin ◽  
Thomas G. Pomorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Evolutionarily Conserved ◽  
A Site ◽  
P Type ◽  
Cell Division Control

P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase.

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

scholarly journals cdk1- and cdk2-Mediated Phosphorylation of MyoD Ser200 in Growing C2 Myoblasts: Role in Modulating MyoD Half-Life and Myogenic Activity

1999 ◽  
Vol 19 (4) ◽  
pp. 3167-3176 ◽  
Author(s):  
Magali Kitzmann ◽  
Marie Vandromme ◽  
Valerie Schaeffer ◽  
Gilles Carnac ◽  
Jean-Claude Labbé ◽  
...  
Keyword(s):  
Half Life ◽  
Site Directed Mutagenesis ◽  
Cyclin B ◽  
Wild Type ◽  
E Box ◽  
Integral Role ◽  
Phosphopeptide Mapping

ABSTRACT We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.

scholarly journals Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis

2002 ◽  
Vol 365 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Antonella De LUCA ◽  
Bartolo FAVALORO ◽  
Stefania ANGELUCCI ◽  
Paolo SACCHETTA ◽  
Carmine Di ILIO
Keyword(s):  
Xenopus Laevis ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Structural Instability ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

scholarly journals Molecular Characterization of Ancylostoma ceylanicum Kunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

2004 ◽  
Vol 72 (4) ◽  
pp. 2214-2221 ◽  
Author(s):  
Daniel Chu ◽  
Richard D. Bungiro ◽  
Maureen Ibanez ◽  
Lisa M. Harrison ◽  
Eva Campodonico ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Site Directed Mutagenesis ◽  
Deficiency Anemia ◽  
Growth Delay ◽  
Ancylostoma Ceylanicum ◽  
Reverse Transcription Pcr ◽  
Third Stage ◽  
Kunitz Type

ABSTRACT Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met26 in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.

scholarly journals Structure-function activity of the human sodium-dependent multivitamin transporter: role of His115 and His254

2011 ◽  
Vol 300 (1) ◽  
pp. C97-C104 ◽  
Author(s):  
Abhisek Ghosal ◽  
Hamid M. Said
Keyword(s):  
Structure Function ◽  
Confocal Imaging ◽  
Site Directed Mutagenesis ◽  
Translational Efficiency ◽  
Maximal Velocity ◽  
Intestinal Epithelial ◽  
Sodium Dependent ◽  
Uptake Process ◽  
Function Activity

Intestinal absorption of biotin occurs via a Na+-dependent carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT; product of the Slc5a6 gene). The SMVT system is exclusively expressed at the apical membrane domain of the polarized intestinal epithelial cells. Whereas previous studies from our laboratory and others have characterized different physiological and biological aspects of SMVT, little is currently known about its structure-function activity relationship. Using site-directed mutagenesis approach, we examined the role of the positively charged histidine (His) residues of the human SMVT (hSMVT) in transporting the negatively charged biotin. Of the seven conserved (across species) His residues in the hSMVT polypeptide, only His115 and His254 were found to be important for the function of hSMVT as their mutation led to a significant reduction in carrier-mediated biotin uptake. This inhibition was mediated via a significant reduction in the maximal velocity ( Vmax), but not the apparent Michaelis constant ( Km), of the biotin uptake process and was not related to the charge of the His residue. The inhibition was also not due to changes in transcriptional or translational efficiency of the mutated hSMVT compared with wild-type carrier. However, surface biotinylation assay showed a significant reduction in the level of expression of the mutated hSMVT at the cell surface, a finding that was further confirmed by confocal imaging. Our results show important role for His115 and His254 residues in hSMVT function, which is most probably mediated via an effect on level of hSMVT expression at the cell membrane.

Effect of matrix metallopeptidase 13 (MMP13) on multiple myeloma (MM) cells, osteoclast (OCL) activity, and bone resorption.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8099-8099
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Rentian Feng ◽  
Mei Hua Jin ◽  
Farideh Sabeh ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Bone Resorption ◽  
Bone Disease ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Bone Lesions ◽  
Lytic Lesions

8099 Background: MM cells produce OCL-activating factors that induce excessive bone resorption resulting in lytic lesions. The role of MMPs in invasion/progression of solid tumors is well-known, but its function in MM has not been well elucidated. Our group has shown that MMP13 is highly expressed in primary MM cells and in sera of MM patients. Levels of MMP13 significantly correlate with the extent of bone disease. MMP13 is induced by IL-6 via AP-1 activation in MM cells and enhances fusion of OCL precursors resulting in excessive bone resorption. OCL formation using MNCs of mmp-13-/- mice resulted in a fusion defect, significantly decreased OCL size and activity, which could be reversed by exogenous MMP13 (ASH 2009, IMW 2011). Methods: Methods will be presented in the Results section. Results: RT-PCR and western blotting revealed that IL-6 treatment of MM cells induced MMP13 transcription (30-fold) and secretion (>1000-fold). Protein expression of the AP-1 members c-Jun and c-Fos was induced by IL-6, which correlated with MMP13 upregulation. Our data further indicate that the catalytic activity of MMP13 is not required to enhance OCL formation and bone resorption. To prove this, we generated the MMP13 activity-dead mutation MMP13-E223A construct by site-directed mutagenesis PCR-based cloning. The mutated protein was overexpressed in HEK293 cells and purified from the supernatant to confirm whether loss of catalytic activity blocks MMP13 function. To further investigate the in vivo role of MMP13 in MM bone disease, MMP13 expression was knocked down (KD) in murine 5TGM1-MM cells by pKLO. 1 puro lentiviral infection containing sh-RNA targeting mouse MMP13 sequence. MMP13-KD 5TGM1-MM cells or WT-5TGM1-MM cells were intratibially injected into RAG2-/- mice. Development of lytic bone lesions are monitored by micro-QCT and data will be available at the time of presentation. Conclusions: Our data suggest that MMP13, secreted by MM cells, plays a critical role in the development of lytic lesions. Targeting MMP13 represents a promising approach to treat or to prevent bone disease in MM.

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